This project is using a combination of methods to analyze the ion transport properties of lysosomal membranes. Lysosomes are intracellular organelles that serve in most cells as digestive organelles although in some tissues they are used for other functions. Disorders of lysosome function lead to a variety of diseases including neurological dysfunction (lysosomal storage diseases) and osteopetrosis (overcalcification of bone). Lysosomes utilize an ATP-driven proton pump to maintain an acidic luminal pH and facilitate their digestive function. Such a pump can only be effective if accompanied by additional ion transport to dissipate the transmembrane voltage built up by the ATPase. Genetic evidence suggests that this additional ion pathway a ClC-type anion transporter, but functional experiments have not yet demonstrated such a pathway. We identified the ion transport pathways in lysosomes, characterized their properties, and to identified the responsible proteins. Last year we began examining endosomes to determine the effects of Cl transport on their acidification processes and began constructing a ClC-7 knockout mouse to further understand the effects of this transporter. In the past year we have focused our efforts on developing methods to measure pH in lysosomes in living cultured cells to examine the influence of ClC-7 on these organelles. We have also continued to develop our knockout mice. A recent publication suggested that a cation flux may also be important in lysosomal acidification and we are using our methods to test this hypothesis.